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Funder: Biotechnology and Biological Sciences Research Council; Id: http://dx.doi.org/10.13039/501100000268, Funder: Wellcome Trust; Id: http://dx.doi.org/10.13039/100010269, Xylan is the most abundant non-cellulosic polysaccharide in grass cell walls, and it has important structural roles. The name glucuronoarabinoxylan (GAX) is used to describe this variable hemicellulose. It has a linear backbone of β-1,4-xylose (Xyl) residues that may be substituted with α-1,2-linked (4-O-methyl)-glucuronic acid (GlcA), α-1,3-linked arabinofuranose (Araf), and sometimes acetylation at the O-2 and/or O-3 positions. The role of these substitutions remains unclear, although there is increasing evidence that they affect the way xylan interacts with other cell wall components, particularly cellulose and lignin. Here, we used substitution-dependent endo-xylanase enzymes to investigate the variability of xylan substitution in grass culm cell walls. We show that there are at least three different types of xylan: (i) an arabinoxylan with evenly distributed Araf substitutions without GlcA (AXe); (ii) a glucuronoarabinoxylan with clustered GlcA modifications (GAXc); and (iii) a highly substituted glucuronoarabinoxylan (hsGAX). Immunolocalization of AXe and GAXc in Brachypodium distachyon culms revealed that these xylan types are not restricted to a few cell types but are instead widely detected in Brachypodium cell walls. We hypothesize that there are functionally specialized xylan types within the grass cell wall. The even substitutions of AXe may permit folding and binding on the surface of cellulose fibrils, whereas the more complex substitutions of the other xylans may support a role in the matrix and interaction with other cell wall components.

The intricate architecture of cell walls and the complex cross-linking of their components hinders some industrial and agricultural applications of plant biomass. Xylan is a key structural element of grass cell walls, closely interacting with other cell wall components such as cellulose and lignin. The main branching points of grass xylan, 3-linked l-arabinosyl substitutions, can be modified by ferulic acid (a hydroxycinnamic acid), which cross-links xylan to other xylan chains and lignin. XAX1 (Xylosyl arabinosyl substitution of xylan 1), a rice (Oryza sativa) member of the glycosyltransferase family GT61, has been described to add xylosyl residues to arabinosyl substitutions modified by ferulic acid. In this study, we characterize hydroxycinnamic acid-decorated arabinosyl substitutions present on rice xylan and their cross-linking, in order to decipher the role of XAX1 in xylan synthesis. Our results show a general reduction of hydroxycinnamic acid-modified 3-linked arabinosyl substitutions in xax1 mutant rice regardless of their modification with a xylosyl residue. Moreover, structures resembling the direct cross-link between xylan and lignin (ferulated arabinosyl substitutions bound to lignin monomers and dimers), together with diferulates known to cross-link xylan, are strongly reduced in xax1. Interestingly, apart from feruloyl and p-coumaroyl modifications on arabinose, putative caffeoyl and oxalyl modifications were characterized, which were also reduced in xax1. Our results suggest an alternative function of XAX1 in the transfer of hydroxycinnamic acid-modified arabinosyl substitutions to xylan, rather than xylosyl transfer to arabinosyl substitutions. Ultimately, XAX1 plays a fundamental role in cross-linking, providing a potential target for the improvement of use of grass biomass.

Additional file 12: Fig. S7. Characterisation of the recombinant bacterial CAZymes encoded by the endosymbionts. DNS reducing sugars assays showing activities on a number of substrates for LpsGH5_8, LpsGH11, LpsGH134a and LpsGH134b. CMC = carboxymethyl cellulose, LBG = locust bean gum. The nanomoles of sugars released by the different proteins cannot be compared quantitatively as different amounts were used for the assay. File format .DOCX.

Additional file 8: Table S2. CAZy families. List of the CAZy families mentioned in the results, with the description of the activities that have been recorded for enzymes listed in each family. The information has been gathered from the CAZy database ( http://www.cazy.org ). File format .DOCX.

Additional file 2: Fig. S2. Searching for the opening of the duct of Deshayes in the shipworm digestive system. Left, transverse sections through the shell valve, from the anterior to posterior adductor muscle (A-E). Right, 3D rendered model of the whole shipworm, with the green highlighted region showing the position of the transverse cross section displayed on the left. File format .DOCX.

Additional file 6: Fig. S6. Immunogold labelling of LpsGH5_8 in L. pedicellatus using pre-immune serum (negative control). A) SEM image illustrating the anatomical position of the gills, food groove, caecum and mouth in the shipworm body. B-D) Immunogold labelling of the lumen of the food groove (B), lumen of the caecum (C), and the gills (D) performed with pre-immune serum. No gold particles are observed in any of the samples. B = bacteria. File format .DOCX.

Additional file 7: Table S1. Details of the proteins identified through proteomic analysis of the crystalline style. The annotation was performed against the NCBI non-redundant database, the signal peptides were identified with the Signal P4.1 server and the CAZy domains were searched using dbCAN. Many of the contigs were out of frame or not full length (particularly the bacterial ones) and therefore the identification of the signal peptide was not possible. File format .DOCX.

Additional file 4: Fig. S4. Bacterial symbionts in L. pedicellatus. A) TEM image of a gill bacteriocyte. Arrows indicate the numerous rod-shaped bacteria in the cell. B) Close-up view of gill bacteria (TEM). C) Detailed TEM showing a cross section of a gill bacterium and some of its features (periplasmic space and membranes). File format .DOCX.

Additional file 5: Fig. S5. SDS-PAGE and nitrocellulose western blot obtained by loading 0.6 ��g of the recombinant purified bacterial LpsGH5_8 (without the appended CBM). A) Coomassie Brilliant Blue stained SDS-PAGE gel of the purified protein. B) Western blot detection performed with the purified pre-immune serum. C) Western blot detection performed with the purified anti-protein serum. File format .DOCX.

Additional file 3: Fig. S3. Scanning electron microscopy of L. pedicellatus. A dissected specimen with the mantle removed, showing the food groove connecting the gills to the mouth. File format .DOCX.

Additional file 9: Table S3. CAZy families of the caecum. Table showing the relative abundance (calculated from the emPAI score) of eukaryotic and prokaryotic CAZy families identified in the proteomic analysis of the caecum content of L. pedicellatus. File format .DOCX.

Dr Marta Busse-Wicher and Sergio Martinez Cuesta of the University of Cambridge discuss the Data Champions programme. Delivered at the Engaging Researchers with Good Data Management event held at St Catharine's College, Cambridge on the 15th of November 2017

Additional file 5: Figure S4. Expression heat-map for the putative conifer GUX. ExIMAGE feature of the Congenie datanase [30] was used to visualise the expression of Picea abies gene MA_84103g0010 which encodes a homologue of PgGUX. Reads encoding the enzyme are clearly enriched in both late and early wood.

Certain xylanases from family GH10 are highly expressed during secondary wall deposition, but their function is unknown. We carried out functional analyses of the secondary-wall specific PtxtXyn10A in hybrid aspen (Populus tremula × tremuloides). PtxtXyn10A function was analysed by expression studies, overexpression in Arabidopsis protoplasts and by downregulation in aspen. PtxtXyn10A overexpression in Arabidopsis protoplasts resulted in increased xylan endotransglycosylation rather than hydrolysis. In aspen, the enzyme was found to be proteolytically processed to a 68 kDa peptide and residing in cell walls. Its downregulation resulted in a corresponding decrease in xylan endotransglycosylase activity and no change in xylanase activity. This did not alter xylan molecular weight or its branching pattern but affected the cellulose-microfibril angle in wood fibres, increased primary growth (stem elongation, leaf formation and enlargement) and reduced the tendency to form tension wood. Transcriptomes of transgenic plants showed downregulation of tension wood related genes and changes in stress-responsive genes. The data indicate that PtxtXyn10A acts as a xylan endotransglycosylase and its main function is to release tensional stresses arising during secondary wall deposition. Furthermore, they suggest that regulation of stresses in secondary walls plays a vital role in plant development.

Additional file 7: Table S1. Average ethanol production during Simultaneous saccharification and Fermentation experiments. This data was used to generate Fig. 1e.

Additional file 3: Figure S3. Sugar release from WT and gux1/2 biomass for saccharification reactions supplemented with glucuronidase GH115. Reaction scheme for GH115 supplementation (A) and D-Xylose release from Col0 (WT) and gux1/2/3 biomass following supplementation (+) or not (-) with GH115 (B). Sterilisation steps were carried out between different stages of the experiment to avoid microbial growth in the reaction tubes. Reactions were performed in triplicate, and xylose measured after stage 1 or stage 3.

Additional file 2: Figure S2. Analysis of gux1/2 plant growth and biomass production. Plant growth is not affected by removal of GlcA branches from xylan. The graphs represent average height of 7 week old plants (A, n = 36 for WT and 34 for gux1/2), average total plant mass (B, n = 36 for WT and 34 for gux1/2) and average mass of 5 cm basal stem sections (C, n = 36 for WT and 33 for gux1/2). Error bars represent standard deviation. There is no statistically significant difference between the values measured for the WT and the mutant plant (Student’s t test).

Additional file 1: Figure S1. Analysis of gxm 1/2/3 plants lacking methylation of GlcA. The triple mutant plants grow to the same height (A). The xylan (B) and GlcA (C) content of gxm1/2/3 plants is not different when compared to WT. Growth images and xylan quantitation are representable for 3 biological replicates.

Additional file 3: Figure S3. Sugar release from WT and gux1/2 biomass for saccharification reactions supplemented with glucuronidase GH115. Reaction scheme for GH115 supplementation (A) and D-Xylose release from Col0 (WT) and gux1/2/3 biomass following supplementation (+) or not (-) with GH115 (B). Sterilisation steps were carried out between different stages of the experiment to avoid microbial growth in the reaction tubes. Reactions were performed in triplicate, and xylose measured after stage 1 or stage 3.

Additional file 4: Table S2. Gene Bank and OneKP transcript catalogue numbers. Those transcripts encode conifer GUX enzymes used to construct the maximum likelihood phylogeny presented in Fig. 2a.

Background Plant lignocellulosic biomass can be a source of fermentable sugars for the production of second generation biofuels and biochemicals. The recalcitrance of this plant material is one of the major obstacles in its conversion into sugars. Biomass is primarily composed of secondary cell walls, which is made of cellulose, hemicelluloses and lignin. Xylan, a hemicellulose, binds to the cellulose microfibril and is hypothesised to form an interface between lignin and cellulose. Both softwood and hardwood xylan carry glucuronic acid side branches. As xylan branching may be important for biomass recalcitrance and softwood is an abundant, non-food competing, source of biomass it is important to investigate how conifer xylan is synthesised. Results Here, we show using Arabidopsis gux mutant biomass that removal of glucuronosyl substitutions of xylan can allow 30% more glucose and over 700% more xylose to be released during saccharification. Ethanol yields obtained through enzymatic saccharification and fermentation of gux biomass were double those obtained for non-mutant material. Our analysis of additional xylan branching mutants demonstrates that absence of GlcA is unique in conferring the reduced recalcitrance phenotype. As in hardwoods, conifer xylan is branched with GlcA. We use transcriptomic analysis to identify conifer enzymes that might be responsible for addition of GlcA branches onto xylan in industrially important softwood. Using a combination of in vitro and in vivo activity assays, we demonstrate that a white spruce (Picea glauca) gene, PgGUX, encodes an active glucuronosyl transferase. Glucuronic acid introduced by PgGUX reduces the sugar release of Arabidopsis gux mutant biomass to wild-type levels indicating that it can fulfil the same biological function as native glucuronosylation. Conclusion Removal of glucuronic acid from xylan results in the largest increase in release of fermentable sugars from Arabidopsis plants that grow to the wild-type size. Additionally, plant material used in this work did not undergo any chemical pretreatment, and thus increased monosaccharide release from gux biomass can be achieved without the use of environmentally hazardous chemical pretreatment procedures. Therefore, the identification of a gymnosperm enzyme, likely to be responsible for softwood xylan glucuronosylation, provides a mutagenesis target for genetically improved forestry trees. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0902-1) contains supplementary material, which is available to authorized users.

Additional file 1: Figure S1. Analysis of gxm 1/2/3 plants lacking methylation of GlcA. The triple mutant plants grow to the same height (A). The xylan (B) and GlcA (C) content of gxm1/2/3 plants is not different when compared to WT. Growth images and xylan quantitation are representable for 3 biological replicates.

Additional file 2: Figure S2. Analysis of gux1/2 plant growth and biomass production. Plant growth is not affected by removal of GlcA branches from xylan. The graphs represent average height of 7 week old plants (A, n = 36 for WT and 34 for gux1/2), average total plant mass (B, n = 36 for WT and 34 for gux1/2) and average mass of 5 cm basal stem sections (C, n = 36 for WT and 33 for gux1/2). Error bars represent standard deviation. There is no statistically significant difference between the values measured for the WT and the mutant plant (Student’s t test).

Additional file 4: Table S2. Gene Bank and OneKP transcript catalogue numbers. Those transcripts encode conifer GUX enzymes used to construct the maximum likelihood phylogeny presented in Fig. 2a.

Additional file 6: Figure S5. Western Blot analysis of N. benthamiana membrane fraction extracted from leaves enriched for GTL6 and PgGUX. PageRulerâ ˘ Prestained Protein Ladder, 10 to 180Â kDa (Thermo-Fisher Scientific) was used as a molecular size marker.

Additional file 7: Table S1. Average ethanol production during Simultaneous saccharification and Fermentation experiments. This data was used to generate Fig. 1e.

Additional file 6: Figure S5. Western Blot analysis of N. benthamiana membrane fraction extracted from leaves enriched for GTL6 and PgGUX. PageRulerâ ˘ Prestained Protein Ladder, 10 to 180Â kDa (Thermo-Fisher Scientific) was used as a molecular size marker.